Telomeric DNA is found at the extreme ends of chromosomes in humans and most other organisms. See Blackburn, E. H. and J. W. Szostak, Annu. Rev. Biochem 1984, 53, 164-94 and Cech, T. R. Angew. Chem. Int. Ed. 2000, 39, 35-43. Telomeres are characterized by a repeat sequence motif which in humans is 5′-TTAGGG-3. This sequence is repeated tens or even hundreds of times through each end of the double-stranded region of the chromosome and beyond, into a single-stranded extension on the 3′-terminus at each end. The telomeres naturally become shorter with each round of cell division until a critical threshold is reached, after which cellular growth arrest (senescence) occurs. Critically short telomeres have been implicated in a range of aging-related diseases. In addition, maintenance of stable telomere lengths, either through the action of the telomerase enzyme or through alternative mechanisms, is a hallmark of cancer cells. See Lansdorp, P. M., EMBO J. 2009, 28, 2532-40
Given the great interest in telomere biology, methods that permit analysis of the integrity and length of telomeres are widely used in research and diagnostic medicine. One such method involves the in situ hybridization of a fluorescent PNA probe to the telomere. See Baerlocher, G. M., et al., Cytometry 2002, 47, 89-99, and Lansdorp, P. M., et al., Hum. Mol. Gen. 1996, 5, 685-91. The conventional PNA probe is 18 nucleobases (18-mer) in length and is complementary to three contiguous repeats of the human telomere sequence. A fluorescent dye such as Cy3 is attached to the PNA N-terminus, which permits imaging of the telomeres via fluorescence microscopy and quantitative analysis by flow cytometry.
Fewer fluorescently labeled PNA probes are able to hybridize to the telomere, however, as telomeres become shorter or are damaged. As a result, the fluorescent signal observed becomes weaker which hampers intracellular visualization of telomeres and their quantitative analysis under biological conditions that promote telomere shortening.
The conventional choice for using a fluorescently labeled PNA rather than a DNA oligonucleotide probably stems from the observation that PNA's exhibit higher hybridization affinity for complementary DNA targets compared with other synthetic DNA analogues. See Egholm, M et al., Nature 1993, 365, 566-68. It was observed, moreover, that an even higher binding affinity can be obtained by introducing one or more substituent groups into the PNA backbone, particularly when the substituent group is introduced at the gamma carbon (γ-C) atom of the PNA monomer. See Dragulescu-Andrasi, A., et al., J. Am. Chem. Soc. 2006, 128, 10258-67. These “γPNAs” (gamma-PNAs), developed by Ly and coworkers, exhibit a helical pre-organization and improved water solubility compared with standard PNA. See FIG. 1 below and Yeh, J. I., et al., J. Am. Chem. Soc. 2010, 132, 10717-27, and Sahu, B., et. al., J. Org. Chem. 2011, 76, 5614-27.